Methods for diagnosing and monitoring the progression of cancer

ABSTRACT

Methods for measuring c-Met levels in urine and blood samples are provided. Methods for diagnosis and prognosis evaluation for cancer are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national phase application of internationalapplication number PCT/US2006/043654, filed Nov. 8, 2006, which claimsthe benefit of U.S. provisional patent application Nos. 60/780,626,filed Mar. 9, 2006, and 60/734,993, filed Nov. 8, 2005, the disclosuresof which are incorporated herein by reference in their entireties.

FIELD

The present invention relates generally to the field of cancer and morespecifically to cancer diagnosis and prognosis.

BACKGROUND

Many transmembrane proteins are proteolytically released from the cellsurface by a process known as ectodomain shedding. Shedding occurs undernormal physiologic conditions and can be increased in certainpathologies. Among the many receptors for which ectodomain shedding hasbeen demonstrated is c-Met, the hepatocyte growth factor (“HGF”)receptor tyrosine kinase (Komada et al., FEBS Lett 1993; 328:25-9, Wajihet al., Circ Res 2002; 90:46-52). HGF is a pleiotropic heparin-bindingprotein identified and isolated based on observations of its mitogenicactivity on hepatocytes and epithelial cells, and independentlyidentified and isolated based on observations of its ability tostimulate cell motility (scatter factor or SF). (Comoglio et al., SeminCancer Biol 2001; 11:153-65, Funakoshi et al., Clin Chim Acta 2003;327:1-23). HGF is typically produced by cells of mesenchymal origin andacts in a paracrine manner on a variety of cellular targets includingepithelial and endothelial cells, hematopoietic cells, neurons andmelanocytes during embryonic development and throughout adulthood, innormal and pathological processes (Birchmeier et al., Nat Rev Mol CellBiol 2003; 4:915-25). HGF is essential for embryonic development, whereit is involved in somite migration, limb bud and limb skeletal muscleformation, placenta formation (Schmidt et al., Nature 1995; 373:699-702,Uehara et al., Nature 1995:373:702-5) and later in organogenesis(Rosario et al., Trends Cell Biol 2003; 13:328-35), in neuraldevelopment (Birchmeier et al., Trends Cell Biol 1998:8:404-10) and intissue repair and regeneration (Jin et al., J Pharmacol Exp Ther 2003;304:654-60, Huh et al., Proc Natl Acad Sci USA 2004; 101:4477-82). Whilethe role of HGF in adult homeostasis is not yet completely understood, agrowing body of evidence suggests that it is an endogenous tissueprotective factor for several major organs and has potent antifibroticactivity (Liu, Am J Physiol Renal Physiol 2004; 287:F7-16).

The MET oncogene was isolated from a human osteogenic sarcoma cell linethat had been chemically mutagenized in vitro. Transforming activity wasdue to a DNA rearrangement where sequences from the TPR (translocatedpromoter region) locus on chromosome 1 fused to sequences from the METlocus on chromosome 7 (TPR-MET) (Furge et al., Oncogene 2000;19:5582-9). This rearrangement has been found in patients with gastriccarcinoma (Yu et al., Cancer 2000; 88:1801-6). Isolation of thefull-length MET proto-oncogene coding sequence revealed structuralfeatures of a membrane spanning receptor tyrosine kinase (Furge et al.,Oncogene 2000; 19:5582-9). The identification of HGF as the naturalligand for c-Met and the identity of SF and HGF united a collection offindings demonstrating that a single receptor transduced multiplebiological activities including motility, proliferation, survival andbranching morphogenesis (Birchmeier et al., Nat Rev Mol Cell Biol 2003;4:915-25). Activation of the c-Met intrinsic tyrosine kinase (TK)activity was required for all of these activities. Consistent with itsrelationship with HGF, c-Met is widely expressed early in development,deletion of the gene is embryonic lethal in mice, and widespreadexpression persists throughout adulthood (Birchmeier et al., id). BothHGF and c-Met are upregulated after kidney, liver or heart injury,suggestive of a general mechanism of protection against tissue damage,as well as one of tissue repair and regeneration (Matsumoto et al.,Kidney Int 2001; 59:2023-38, Michalopoulos et al., Science 1997;276:60-6, Nakamura et al., J Clin Invest 2000; 106:1511-9).

HGF and c-Met are implicated in a wide variety of human malignanciesincluding colon, gastric, bladder, breast, kidney, liver, lung, head andneck, thyroid and prostate, but also sarcomas, hematologicalmalignancies, melanoma and central nervous system (CNS) tumors(Birchmeier et al., Nat Rev Mol Cell Biol 2003; 4:915-25, Birchmeier etal., Ciba Found Symp 1997; 212:230-40). Through paracrine signaling,overexpression of ligand and/or receptor, autocrine loop formationand/or receptor mutation and gene rearrangement, this signaling pathwaycan enhance tumor cell growth, proliferation, survival, motility andinvasion. Inappropriate c-Met signaling in disease can resemble, atleast in part, developmental transitions between epithelial andmesenchymal cell types normally regulated by HGF. Among the many genesupregulated in response to activation of this pathway is that of thereceptor itself, creating the potential for c-Met overexpression inotherwise normal target cells through persistent ligand stimulation;consistent with this, c-Met overexpression is widely observed in cancersof epithelial origin where paracrine delivery of HGF results indysregulated signaling, whereas cells of mesenchymal origin thatnormally express HGF often acquire c-Met expression, and severalsarcomas display autocrine c-Met signaling (Furge et al., Oncogene 2000;19:5582-9). Importantly, the c-Met pathway activates a program of celldissociation and increased cell motility coupled with increased proteaseproduction that has been shown to promote cellular invasion throughextracellular matrices, and that closely resembles tumor metastasis invivo (Birchmeier et al., Ciba Found Symp 71p 1997; 212:230-40). Inaddition, pathway activation in vascular cells stimulates tumorangiogenesis, facilitating tumor growth for cancers that are growthlimited by hypoxia, and promoting tumor metastasis. Hypoxia aloneupregulates c-Met expression and enhances HGF/SF signaling in culturedcells and mouse tumor models (Pennacchietti et al., Cancer Cell 2003;3:347-61).

Early diagnosis is a key strategy in cancer treatment. Although it isknown that c-Met is overexpressed in certain cancers, a need exists fora screening test for c-Met that is sensitive, cost-efficient, and can beused for diagnosis, determining stage of disease, prognosis, and/orassessing the efficacy of therapeutic intervention. The invention isdirected to this and other ends.

SUMMARY

The present invention generally relates to methods for diagnosing cancerin a patient or methods for identifying a patient having an increasedrisk of developing cancer. The methods provide for analysis of patientsamples to identify the presence of tumor cells in the patient due tothe observation that tumor cells expressing c-Met shed more c-Metectodomain than their normal tissue counterparts, independent of changesin overall c-Met expression levels, and that this shedding is enhancedwith increasingly malignant phenotype. Moreover, the methods fordiagnosis provide that c-Met ectodomain shedding can be quantitated incell lysates, culture supernatants, and biological samples, such asblood and urine, and that c-Met levels are measurable before tumorsbecome palpable yet correlate directly with tumor volume. Thus, it wasconcluded that for a variety of cancers, c-Met ectodomain sheddingprovides a reliable and practical indicator of, among other things,malignant potential and overall tumor burden.

A method for identifying a patient having an increased risk ofdeveloping cancer which comprises the steps of measuring an amount ofsoluble c-Met ectodomain in a biological sample of the patient; anddetecting an increased concentration of soluble c-Met ectodomain in thebiological sample when compared to the concentration of soluble c-Metectodomain in a corresponding biological sample from a controlpopulation, wherein an increased concentration of soluble c-Metectodomain identifies the patient as having an increased risk ofdeveloping cancer. The biological sample includes, but is not limitedto, blood plasma or urine. The concentration of soluble c-Met ectodomainin a urine sample and a blood plasma sample is measured and the combinedmeasurement can be used to determine if the patient has the increasedrisk of developing cancer. An increased concentration of soluble c-Metectodomain can identify the patient as having an increased risk ofdeveloping metastatic cancer.

In one aspect, a soluble c-Met ectodomain level of about 3500 pg c-Metper mg urine creatinine or greater is indicative of an increased risk ofdeveloping cancer in the patient. In a further aspect, a soluble c-Metectodomain level of about 4900 pg c-Met per mg urine creatinine orgreater is indicative of an increased risk of developing cancer in thepatient. In a further aspect, a soluble c-Met ectodomain level of about3800 pg c-Met per mg urine creatinine or greater is indicative of anincreased risk of developing cancer in the patient. In a detailedaspect, a soluble c-Met ectodomain level of about 4200 pg c-Met per mgurine creatinine or greater is indicative of an increased risk ofdeveloping cancer in the patient. In a detailed aspect, a soluble c-Metectodomain level of about 4500 pg c-Met per mg urine creatinine orgreater is indicative of an increased risk of developing cancer in thepatient. The cancer diagnosis can be a cancer of the urinary tract. Thecancer includes, but is not limited to, kidney cancer, renal cancer,prostate cancer, bladder cancer, renal cell carcinoma, or breast cancer.

A method for monitoring the progression of cancer in a patient isprovided which comprises the steps of measuring an amount of solublec-Met ectodomain in a first biological sample at a first time point,measuring an amount of soluble c-Met ectodomain in a second biologicalsample at a second time point, and determining progression of cancer inthe patient based upon a change in amount of soluble c-Met ectodomain inthe first biological sample compared to the second biological sample.The amount of soluble c-Met ectodomain in a urine sample or blood plasmasample, or a combination thereof, is measured and the combinedmeasurement is used to determine the progression of cancer.

A method for measuring the response to cancer therapy in a patient isprovided which comprises the steps of measuring an amount of solublec-Met ectodomain in a biological sample of the patient at a first timepoint, measuring an amount of soluble c-Met ectodomain in a secondbiological sample of the patient at a second time point, and determiningresponse to cancer therapy in the patient based upon the change inamount of soluble c-Met ectodomain in the first biological samplecompared to the second biological sample. In one aspect, a decrease inthe amount of soluble c-Met ectodomain in the first biological samplecompared to the second biological sample indicates a positive responseto cancer therapy in the patient. The cancer therapy can be ananti-cancer drug, for example, a chemotherapeutic agent, ionizingradiation therapy or hormone ablation therapy.

A method of assaying for an amount of c-Met ectodomain present in abiological sample from a human patient, said method is provided whichcomprises assaying the biological sample to determine an amount of c-Metectodomain present in the biological sample from the patient, andcomparing the amount of c-Met ectodomain present in the biologicalsample from the patient to an amount of c-Met ectodomain found in abiological sample of a control population, wherein an increase in theamount of c-Met ectodomain found in the sample of the patient comparedto the amount of c-Met ectodomain found in the sample of the controlpopulation is indicative of the patient having cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. c-Met shedding in a cultured cell model of breast tumorprogression. 1A. Reducing SDS-PAGE and immunoblot analysis of the c-Metexpression in four mammary cell lines; p145 is the intact c-Met betasubunit. In this model, M2, M3 and M4 represent derivatives of thenormal breast cell line M1 with increasingly malignant phenotype. Thenormal mammary epithelial cell line B5/589 was used as a positivecontrol for c-Met expression, and lysates were immunoblotted for betaactin to confirm equal sample loading. 1B. Reducing SDS-PAGE andimmunoblot analysis of c-Met ectodomain shedding by cell lines M1, M2,M3 and M4. Culture supernatant from B5/589, as well as a purifiedrecombinant c-Met ectodomain IgG Fc fusion protein (358-MT, R&D Systems)were used as positive controls of c-Met shedding and ectodomainrecognition, respectively. Molecular masses of predominant c-Metectodomain fragments are indicated in kDa at left.

FIG. 2. Quantitation of c-Met shedding using an electrochemiluminescenttwo site immunoassay. 2A. Standard curve of purified recombinant c-Metectodomain-IgG fusion protein (358-MT). Mean values from quadruplicatesamples are shown as log[soluble c-Met] in ng/ml vs. log[signalintensity] in relative units. Standard deviations are smaller than thesymbol size. 2B. Analysis of sample quenching in a431 human epidermoidcarcinoma cells. Cells were serum deprived for 24 h and soluble c-Metectodomain was measured in the presence and absence of added 358-MT.Mean values of quadruplicate samples from 358-MT (358MT, open bar), A431conditioned medium (A431, dark gray bar), and A431 conditionedmedium+358-MT (obs, light gray bar) are shown +/−standard deviation. Theexpected sum of 358-MT and A431 conditioned medium sample signals isalso shown (exp, dark-gray/open bar). 2C. Soluble c-Met in 24 h B5/589conditioned media (B5, open bar) was filtered using a 10 kDa cutoffmolecular sieve and soluble c-Met in the filtrate was measured (YM10,dark gray bar). 24 h B5/589 conditioned medium (open bar) wasimmunodepleted using an ectodomain-specific monoclonal antibody (mAbDO24, Upstate Biotechnology; light gray bar). 2D. HGF binding (ng/ml)was measured in samples containing equivalent amounts of a c-Met Fcfusion protein (open squares) and c-Met ectodomain present in B5/589conditioned medium (black circles), as a function of the concentrationof HGF added (ng/ml). Error bars represent standard deviation; where nobars are seen, the deviation is smaller than the symbol size.

FIG. 3. Quantitation of c-Met expression and shedding in a cultured cellmodel of breast tumor progression. Cell surface and soluble c-Met levelswere measured in the MCF10A derived cell lines M1 (open bars), M2 (lightgray bars), M3 (dark gray bars) and M4 (black bars). Non-ionic detergentextractable c-Met is expressed as pg/ug cell protein (Cellular c-Met,top panel); c-Met ectodomain present in 24 h cell conditioned medium isshown expressed as pg/ug cell protein (Soluble c-Met, 2^(nd) panel) andafter normalization to detergent extractable c-Met (Soluble/Cellular,3^(rd) panel). The percentage of detergent extractable c-Met that becamesoluble in 24 h is shown in the bottom panel.

FIG. 4. Quantitation of c-Met expression and shedding in paired cellline models of cancer progression. Cell line pairs, each derived from asingle patient, were compared for levels of cellular (top panel) andsoluble (2^(nd) panel) c-Met. These data were used to calculate theamount of soluble receptor shed after 24 h corrected for cellularreceptor (3^(rd) panel) and the amount of soluble receptor produced in24 h as a percentage of cellular receptor (bottom panel). Cell linesderived from normal tissue (open bars) corresponding to the tissue oftumor origin were paired with tumor-derived cell lines (light gray bars)and other tumor derived cell lines (dark gray bars) were paired withcell lines derived from corresponding metastatic lesions (black bars).CRL7636 is a normal skin cell line; CRL7637 is a skin melanoma cellline; HTB125 is a normal mammary gland cell line; HTB126 is a mammarygland ductal carcinoma cell line; CCL228 is a colorectal adenocarcinomacell line; CCL227 is a lymph node metastasis of colorectaladenocarcinoma cell line; UOK124 is a renal cell carcinoma cell line;and UOK124 LN is a lymph node metastasis of renal cell carcinoma cellline.

FIG. 5. Genetically modified cultured cell models of cancer progression.5A. The human prostate cancer derived cell line PC3 (open bars), aderived metastatic variant PC3M (black bars) and PC3M transfected withan ADxA gene expression plasmid (+MxA, gray bars) were compared forlevels of cellular (top panel) and soluble (2^(nd) panel) c-Met. Thesedata were used to calculate the amount of soluble receptor shed after 24h corrected for cellular receptor (3^(rd) panel) and the amount ofsoluble receptor produced in 24 h as a percentage of cellular receptor(bottom panel). 5B. The human breast cancer derived cell line C100(black bars) and a C100 derivative cell line cloned after transfectionwith an NM23 gene expression plasmid (H1.177, gray bars) were comparedfor levels of cellular and soluble c-Met as in Panel A.

FIG. 6. c-Met shedding in mice bearing human tumor cell xenografts.Soluble human c-Met concentrations in plasma (ng/ml, upper panels) orurine samples (pg/ml, lower panels) obtained from mice bearingsubcutaneous UOK261 human bladder carcinoma (left panels) or U-87 MGhuman glioblastoma (right panels) cell xenografts plotted against thecorresponding tumor volume (mm³) in each mouse at the time of sampleprocurement. Values represent the mean of triplicate measurements;standard deviations are smaller than the symbol size. Each line andcorresponding R² value represent a best fit linear regression analysisforced through the origin and were performed using GraphPad Prismsoftware.

FIG. 7. Creatinine normalized c-Met values of human patient urinesamples. FIG. 7 provides a column scattergram illustrating the c-Metvalues in human patient urine sample. The Y axis shows c-Met pg/mgcreatinine. The following acronyms are used on the X (Category) axis:HLRCC refers to Hereditary Leiomyomatosis and Renal Cell Carcinoma(inherited kidney cancer syndrome); BPH refers to Benign ProstaticHypertrophy (possibly prelude to prostate cancer); CaP refers toProstate Cancer; CaB refers to Bladder Cancer; and PRC refers toPapillary Renal Cancer, sporadic, not inherited. Each data point isshown as a symbol. The horizontal line represents the median.Statistical analysis was not performed for this data set.

FIG. 8. Creatinine normalized c-Met mean values of human patient urinesamples. FIG. 8 provides a bar graph illustrating the mean values ofc-Met in the patient populations. The Y axis shows c-Met pg/mgcreatinine. The following acronyms are used on the X (Category) axis:HLRCC refers to Hereditary Leiomyomatosis and Renal Cell Carcinoma(inherited kidney cancer syndrome); BPH refers to Benign ProstaticHypertrophy (possibly prelude to prostate cancer); CaP refers toProstate Cancer; CaB refers to Bladder Cancer; and PRC refers toPapillary Renal Cancer, sporadic, not inherited. Statistical analysiswas not performed for this data set.

FIG. 9. Creatinine normalized c-Met values over “No Malignancy” group.FIG. 9 provides a bar graph illustrating normalized c-Met values over“No Maligancy group”. The Y shows fold increase in c-Met. The followingacronyms are used on the X (Category) axis: HLRCC refers to HereditaryLeiomyomatosis and Renal Cell Carcinoma (inherited kidney cancersyndrome); BPH refers to Benign Prostatic Hypertrophy (possibly preludeto prostate cancer); CaP refers to Prostate Cancer; CaB refers toBladder Cancer; and PRC refers to Papillary Renal Cancer, sporadic, notinherited. Statistical analysis was not performed for this data set.

FIG. 10: Sequence for c-Met extracellular domain. FIG. 10 provides thesequence for the c-met extracellular domain, SEQ ID NO: 1 showing thefunctional domains. The c-Met ectodomain comprises residues 25 to 932 ofthe c-Met extracellular domain.

FIG. 11: c-Met full length sequence. FIG. 11 provides the full lengthc-Met sequence, SEQ ID NO:2.

FIG. 12: c-Met in normal patients versus patients with bladder cancer.FIG. 12 provides a box and whisker graph illustrating c-Met shedding ina group of normal patients versus patients with bladder cancer. The boxextends from the 25^(th) percentile to the 75^(th) percentile, with ahorizontal line at the median (50^(th) percentile). Whiskers extend downto the smallest value and up the largest.

FIG. 13: Immunodepletion of plasma samples.

DETAILED DESCRIPTION Overview

The present inventors have observed, after analyzing patient samples,that tumor cells expressing c-Met shed more c-Met ectodomain than theirnormal tissue counterparts, independent of changes in overall c-Metexpression levels, and that this shedding is enhanced with increasinglymalignant phenotype. Moreover, the present inventors have observed,after further analysis, that c-Met ectodomain shedding can bequantitated in cell lysates, culture supernatants, and biologicalsamples, such as blood and urine, and that c-Met levels are measurablebefore tumors become palpable yet correlate directly with tumor volume.Thus, it was concluded that for a variety of cancers, c-Met ectodomainshedding provides a reliable and practical indicator of, among otherthings, malignant potential and overall tumor burden. The observationand conclusion that c-Met can be detected in urine and provides areliable and practical indicator of malignant potential and overalltumor burden was particularly surprising given the large size of thec-Met ectodomain, the low expression levels of c-Met, the acidic pH ofurine, and the protease content in urine.

This invention provides, inter alia, methods and assays for diagnosingcancer in a subject, identifying a patient having an increased risk ofdeveloping cancer, monitoring the progression of cancer in a subject,measuring a clinical parameter related to cancer in subject, andmeasuring the therapeutic response to a cancer drug. In a particularlypreferred embodiment, the present invention provides methods and assaysfor diagnosing bladder cancer in a subject, monitoring the progressionof bladder cancer in a subject, measuring a clinical parameter relatedto bladder cancer in subject, and measuring the therapeutic response toa cancer drug for the treatment of bladder cancer.

“Cancer” refers to any of a number of diseases that are characterized byuncontrolled, abnormal proliferation of cells, as well as any of anumber of characteristic structural and/or molecular features. A“cancerous cell” is understood as a cell having specific structuralproperties, lacking differentiation and in many instances, being capableof invasion and metastasis, see DeVita, V. et al. (eds.), 2001, CancerPrinciples and Practice of Oncology, 6th. Ed., Lippincott Williams &Wilkins, Philadelphia, Pa.). The term cancer includes, for example,cancers of the female reproductive organs including, for example,ovarian cancer, cervical cancer and uterine cancer; lung cancer; breastcancer; renal cell carcinoma; Hodgkin's lymphoma; Non-Hodgkin'slymphoma; cancers of the genitourinary system including, for example,kidney cancer, prostate cancer, bladder cancer, and urethral cancer;cancers of the head and neck; liver cancer; cancers of thegastrointestinal system including, for example, stomach cancer,esophageal cancer, small bowel cancer or colon cancer; cancers of thebiliary tree; pancreatic cancer; cancers of the male reproductive systemincluding, for example, testicular cancer; Gestational trophoblasticdisease; cancers of the endocrine system including, for example, thyroidcancer, parathyroid cancer, adrenal gland cancer, carcinoid tumors,insulinomas and PNET tumors; sarcomas, including, for example, Ewing'ssarcoma, osteosarcoma, liposarcoma, leiomyosarcoma, andrhabdomyosarcoma; mesotheliomas; cancers of the skin; melanomas; cancersof the central nervous system; pediatric cancers; and cancers of thehematopoietic system including, for example, all forms of leukemia,myelodysplastic syndromes, myeloproliferative disorders and multiplemyeloma. Cancers referred to in the present methods include thosewherein the c-Met signaling pathway is overexpressed, accompanied byincreased protease production and/or contributes to malignancy.Exemplary cancers include, for example urological cancers, such asbladder cancer; carcinomas, such as bladder, breast, cervical,cholangiocarcinoma, colorectal, oesophageal, gastric, head and neck,kidney, liver, lung, nasopharyngeal, ovarian, pancreas/gall bladder,prostrate and thyroid carcinomas; musculoskeletal carcinomas, such as,osteosarcoma, synovial sarcoma, and rhabdomyosarcoma; soft tissuesarcomas, such as, MFH/fibrosarcoma, leiomyosarcoma, and kaposi'ssarcoma; haematopietic malignancies, such as, multiple myeloma,lymphomas, adult T-cell leukemia, acute myelogenous leukemia, andchronic myeloid leukemia; and other neoplasms, such as glioblastomas,astrocytomas, melanoma, mesothelioma, and Wilms' tumor (Birchmeier etal., Nat Rev Mol Cell Bio 2003 4(12):912-925.

In certain embodiments, the present invention provides methods ofdiagnosing cancer in a patient or methods for identifying a patienthaving an increased risk of developing cancer. Diagnosis, as usedherein, includes not only the initial identification of a cancerassociated with c-met ectodomain shedding in a patient but confirmatorytesting, or screening in patients who have previously been identified ashaving or likely to have a cancer associated with c-met ectodomainshedding. The methods include the steps of providing a biological samplefrom the patient, measuring the amount of soluble c-Met ectodomain inthe biological sample, preferably a urine and/or blood plasma sample,and determining if the patient has a greater likelihood of cancer basedon the amount of c-Met measured. A patient has a greater likelihood ofhaving cancer when he has a c-Met level that is greater than the meanc-Met level in a non-cancerous population.

In certain embodiments, a patient having a greater likelihood of cancerwill have an amount of c-Met present in a biological sample, preferablya urine and/or plasma sample, that is at least two times the mean amountof c-Met present in a corresponding sample from a non-cancerous controlpopulation, at least three times the mean amount of c-Met present in acorresponding sample from a non-cancerous control population, at leastfour times the mean amount of c-Met present in a corresponding samplefrom a non-cancerous control population, or at least seven times themean amount of c-Met present in a corresponding sample from anon-cancerous control population.

The present inventors have analyzed patient samples and after analysisconcluded that the mean c-Met levels in a non-cancerous controlpopulation (n=30) is 4193 mg c-Met per mg urine creatinine with astandard error of 335. Further statistical analysis has shown that thelower 95% confidence level is 3508 mg c-Met per mg urine creatinine andthe higher 95% confidence level is 4878 mg c-Met per mg urinecreatinine. Accordingly, in certain embodiments of the presentinvention, a measurement of c-Met of about 3500 or greater, about 3800or greater, about 4200 or greater, about 4500 or greater, or about 4900c-Met per mg urine creatinine or greater, in a urine sample from apatient will be indicative of a greater likelihood of cancer in thepatient. Similarly, a measurement of c-Met of less than about 3500 c-Metper mg urine creatinine will indicate that the patient has a smallerrisk than the general population of having cancer.

The present inventors have analyzed the patient data and concluded thatthe mean c-Met levels in a population (n=12) having bladder cancer is10,600 mg c-Met per mg urine creatinine with a standard error of 2678.Further statistical analysis has shown that the lower 95% confidencelevel is 4709 mg c-Met per mg urine creatinine and the higher lower 95%confidence level is 16,500 mg c-Met per mg urine creatinine.Accordingly, in certain embodiments of the present invention, ameasurement of c-Met of about 4700 or greater, about 6000 or greater,about 7000 or greater or about 8000 or greater, c-Met per mg urinecreatinine or greater, in a urine sample from a patient, will beindicative of a greater likelihood of having cancer, in particularbladder cancer

A human sample value, obtained in triplicate or greater replicates, witha mean above the upper limit of the normal range (defined by the upper95% Confidence Interval, or 4878 pg c-Met/mg Creatinine), and determinedto be significantly different (as defined using the Student's t-test orother appropriate statistical analysis) from the normal mean presentedherein (4193 pg c-Met/mg Creatinine), indicates a greater likelihood ofhaving cancer or other hyperplastic disease, in particular bladdercancer.

A patient having a greater likelihood of having cancer can then befurther screened to determine whether the subject does, in fact, havecancer. These screening methods include, for example, conventionalwork-ups to determine risk factors that are associated with cancer, ahistory and physical or neurological exam with particular attention toobvious lesions; palpable masses; ulcerations; swelling or enlargementof any masses or organs; erosion of bone; laterality; size and number ofpalpable lymph nodes; vision changes, focal deficit, tumor impingementon a specific nerve or structure; evidence of increased intracranialpressure; evidence of obstructive hydrocephalus. A diagnosis of cancercan be confirmed, for example, by imaging tests such as X-rays, nuclearscans and/or biopsies.

The methods of the invention rely, in part, on measuring the amount ofc-Met in a sample. The c-met protooncogene is transmembrane protein thatis derived from a 170 kDa precursor. The mature c-met protein iscomposed of a 50 KDa α subunit that is linked by 2 disulfide bonds to a145 kDA β subunit. The c-Met ectodomain comprises residues 25 to 932ofthe c-Met extracellular domain, SEQ ID NO: 1, as shown in FIG. 11.

As used herein, the phrase “measuring the amount of c-Met or solublec-Met ectodomain” means any direct or indirect quantitative assay forc-Met ectodomain, including fragments thereof.

Biological sample refers to a biological tissue or biological fluid froma patient or human patient. The biological tissue can be assayed forc-Met ectodomain as a detergent extract of the tissue. Biological tissuefrom a patient can be extracted with non-ionic detergent buffersolutions as described herein for preparing cell extracts. The tissuecan be minced or ground (at 4° C. or on ice) to maximize surface areaavailable for extraction, and generally the extraction (also on ice) isperformed for a much longer period (2-3 hours) as opposed to a fewminutes for cultured cells.

A variety of biological samples can be useful in practicing the methodsof the invention including, for example, blood, serum, plasma, urine,salivary fluid, ascite fluid and the like. A preferred biological sampleis urine.

Such assays provide valuable means of monitoring the status ofneoplastic diseases. In addition to improving prognostication, knowledgeof the disease status allows the attending physician to select the mostappropriate therapy for the individual patient. For example, patientswith a high likelihood of relapse can be treated rigorously, usuallyinvolving systemic chemotherapy and/or radiation therapy. When there isa lesser likelihood of relapse, less aggressive therapies can be chosen.Because of the severe patient distress caused by the more aggressivetherapy regimens, it would be desirable to distinguish with a highdegree of certainty those patients requiring such aggressive therapies.See for example, U.S. Pat. No. 5,674,753, incorporated herein byreference in its entirety.

The present methods are useful for screening a wide variety ofneoplastic diseases, including both solid tumors and hematopoieticcancers. Exemplary neoplastic diseases include carcinomas, such asadenocarcinomas and melanomas, and squamous cell carcinomas of the headand neck; mesodermal tumors, such as neuroblastomas and retinoblastomas;sarcomas, such as osteosarcomas, Ewing's sarcoma, and various leukemias;and lymphomas. Of particular interest are gynecologic cancers includingovarian, cervical, vaginal, endometrial and vulval cancers;gastrointestinal cancer, such as, stomach, colon and esophageal cancers;urinary tract cancer, such as, bladder and kidney cancers; skin cancer;liver cancer; prostate cancer; lung cancer; and breast cancer. Of stillfurther particular interest are gynecologic cancers; breast cancer;urinary tract cancers, especially bladder cancer; lung cancer;gastrointestinal cancer, such as, stomach, colon and esophageal cancers;and liver cancer. Even further of particular interest are gynecologiccancers and breast cancer. Tumors which are known to overexpress intactc-Met may be good candidates for target neoplastic diseases for theassays of this invention, that is, such tumors may release into bodyfluids the c-Met ectodomain protein at elevated levels above normal.Particularly well studied are tumors of the breast and adenocarcinomasof the vulva which have been confirmed to overexpress c-Met. Preferably,in regard to tumors of the lung and cervix, squamous cell carcinomashave been particularly associated with elevated levels of c-Met. c-Metmeasurements have been determined to be markers for breast cancer and toprovide valuable information diagnostically and prognostically, incorrelation with other markers, such as, hormone receptor measurements,for example, with estrogen receptors (ERs) and progesterone receptors(PRs).

The body fluids that are of particular interest in assaying for thec-Met ectodomain protein according to the methods of this inventioninclude blood, serum, semen, breast exudate, saliva, sputum, urine,cytosols, plasma, ascites, pleural effusions, amniotic fluid, bladderwashes, bronchioalveolar lavages, and cerebrospinal fluid. Blood, serum,plasma, and urine are useful for screening according to the methods ofthis invention. The assays of this invention may also be useful indetecting and/or quantitating said c-Met ectodomain protein in tissueextracts.

From a knowledge of the structure of the external domain of the c-Met, anumber of monoclonal or polyclonal antibodies can be generated thatspecifically recognize that protein. Because said c-Met ectodomainprotein is found to exist freely in the biological fluids of mammals, itis possible to detect and/or quantitate the levels of that protein.Utilizing current antibody detection techniques that can quantitate thebinding of monoclonal antibodies, made specifically to epitopes on theexternal domain of the c-Met, in particular to epitopes on theN-terminal, one can determine the amount of said c-Met ectodomainprotein in the fluids of cancer patients.

Such an assay can be used to detect tumors, quantitate their growth, andhelp in the diagnosis and prognosis of the human disease. The assays canalso be used to detect the presence of cancer metastasis, as well asconfirm the absence or removal of all tumor tissue following surgery,cancer chemotherapy and/or radiation therapy. It can further be used tomonitor cancer chemotherapy and tumor reappearance. Representativeimmunoassays involve the use of monoclonal or polyclonal antibodieswhich can be appropriately labeled to detect and/or quantitate saidc-Met ectodomain protein in the body fluids of mammals.

Diagnosis is based on analyzing the sample for amount of c-Met andcomparing it to a reference value, where the reference value serves toassist in differentiating those with cancer or a certain stage of cancerfrom other individuals. Statistical analysis routines can be performedto develop predictive models for identification of the differentclinical parameters described herein. Measuring an “amount” in a samplemeans quantifying c-Met by determining, for example, the relative orabsolute amount of protein.

A variety of assays for detecting c-Met are known in the art and includedirect and indirect assays for c-Met protein. Immunoassays, includingradioimmunoassays, enzyme-linked immunoassays and two-antibody sandwichassays as described further below, are useful in the methods of theinvention. Furthermore, monoclonal and polyclonal anti-c-Met antibodiesuseful in immunoassays can be readily obtained from a variety ofsources.

A variety of immunoassay formats, including competitive andnon-competitive immunoassay formats, antigen capture assays andtwo-antibody sandwich assays also are useful the methods of theinvention (Self and Cook, Curr. Opin. Biotechnol. 7:60-65 (1996)). Inone embodiment, a method of the invention relies on one or more antigencapture assays. In an antigen capture assay, antibody is bound to asolid phase, and sample is added such that c-Met antigen is bound by theantibody. After unbound proteins are removed by washing, the amount ofbound antigen can be quantitated, if desired, using, for example, aradioassay (Harlow and Lane, Antibodies A Laboratory Manual Cold SpringHarbor Laboratory: New York, 1988)). Immunoassays can be performed underconditions of antibody excess, or as antigen competitions, to quantitatethe amount of antigen and, thus, determine a level of c-Met.

Enzyme-linked immunosorbent assays (ELISAs) can be useful in certainmethods of the invention. An enzyme such as horseradish peroxidase(HRP), alkaline phosphatase (AP), β-galactosidase or urease can belinked, for example, to an anti-c-Met antibody or to a secondaryantibody for use in a method of the invention. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable at 450 nm. Otherconvenient enzyme-linked systems include, for example, the alkalinephosphatase detection system, which can be used with the chromogenicsubstrate p-nitrophenyl phosphate to yield a soluble product readilydetectable at 405 nm. Similarly, a β-galactosidase detection system canbe used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG) to yield a soluble productdetectable at 410 nm, or a urease detection system can be used with asubstrate such as urea-bromocresol purple (Sigma Immunochemicals, St.Louis, Mo.). Useful enzyme-linked primary and secondary antibodies canbe obtained from a number of commercial sources such as JacksonImmuno-Research (West Grove, Pa.) as described further below.

In certain preferred embodiments, c-Met ectodomain is detected andmeasured using chemiluminescent detection. For example in certainembodiments, c-Met ectodomain specific antibodies are used to capturec-Met ectodomain present in the biological sample and a antibodyspecific for the c-Met ectodomain specific antibodies and labeled withan chemiluminescent label is used to detect the c-Met ectodomain presentin the sample. Any chemiluminescent label and detection system can beused in the present methods. Chemiluminescent secondary antibodies canbe obtained commercially from various sources such as Amersham. Methodsof detecting chemiluminescent secondary antibodies are known in the artand are not discussed herein in detail.

Fluorescent detection also can be useful for detecting c-Met in certainmethods of the invention. Useful fluorochromes include, withoutlimitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin,B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red and lissamine.Fluorescein or rhodamine labeled α2-MG-, HA-, TIMP-1- or YKL-40-specificbinding agents such as anti-α2-MG, anti-HA, anti-TIMP-1, or anti-YKL-40antibodies, or fluorescein- or rhodamine-labeled secondary antibodiescan be useful in the invention. Useful fluorescent antibodies can beobtained commercially, for example, from Tago Immunologicals(Burlingame, Calif.) as described further below.

Radioimmunoassays (RIAs) also can be useful in certain methods of theinvention. Such assays are well known in the art. Radioimmunoassays canbe performed, for example, with ¹²⁵I-labeled primary or secondaryantibody (Harlow and Lane, supra, 1988).

A signal from a detectable reagent can be analyzed, for example, using aspectrophotometer to detect color from a chromogenic substrate; aradiation counter to detect radiation, such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. Where an enzyme-linked assayis used, quantitative analysis of the amount of c-Met can be performedusing a spectrophotometer such as an EMAX Microplate Reader (MolecularDevices; Menlo Park, Calif.) in accordance with the manufacturer'sinstructions. The assays of the invention can be automated or performedrobotically, if desired, and that the signal from multiple samples canbe detected simultaneously.

The methods of the invention also encompass the use of capillaryelectrophoresis based immunoassays (CEIA), which can be automated, ifdesired. Immunoassays also can be used in conjunction with laser-inducedfluorescence as described, for example, in Schmalzing and Nashabeh,Electrophoresis 18:2184-93 (1997), and Bao, J. Chromatogr. B. Biomed.Sci. 699:463-80 (1997). Liposome immunoassays, such as flow-injectionliposome immunoassays and liposome immunosensors, also can be used todetect c-Met or to determine a level of c-Met according to certainmethods of the invention (Rongen et al., J. Immunol. Methods 204:105-133(1997)).

Sandwich enzyme immunoassays also can be useful in certain methods ofthe invention. In a two-antibody sandwich assay, a first antibody isbound to a solid support, and the antigen is allowed to bind to thefirst antibody. The amount of c-Met is quantitated by measuring theamount of a second antibody that binds to it.

Quantitative western blotting also can be used to detect c-Met or todetermine a level of c-Met in a method of the invention. Western blotscan be quantitated by well known methods such as scanning densitometry.As an example, protein samples are electrophoresed on 10% SDS-PAGELaemmli gels. Primary murine monoclonal antibodies, for example, againstc-Met are reacted with the blot, and antibody binding confirmed to belinear using a preliminary slot blot experiment. Goat anti-mousehorseradish peroxidase-coupled antibodies (BioRad) are used as thesecondary antibody, and signal detection performed usingchemiluminescence, for example, with the Renaissance chemiluminescencekit (New England Nuclear; Boston, Mass.) according to the manufacturer'sinstructions. Autoradiographs of the blots are analyzed using a scanningdensitometer (Molecular Dynamics; Sunnyvale, Calif.) and normalized to apositive control. Values are reported, for example, as a ratio betweenthe actual value to the positive control (densitometric index). Suchmethods are well known in the art as described, for example, in Parra etal., J. Vasc. Surg. 28:669-675 (1998).

As described herein above, immunoassays including, for example,enzyme-linked immunosorbent assays, radioimmunoassays and quantitativewestern analysis, can be useful in the diagnostic methods of theinvention. Such assays typically rely on one or more antibodies, forexample, anti-c-Met antibodies. As used herein, the term “antibody” isused in its broadest sense to include polyclonal and monoclonalantibodies, as well as polypeptide fragments of antibodies that retainbinding activity for c-Met of at least about 1×10⁵ M⁻¹. One skilled inthe art understands that antibody fragments such as anti-c-Met antibodyfragments and including Fab, F(ab′)₂ and Fv fragments can retain bindingactivity for c-Met and, thus, are included within the definition of theterm antibody as used herein. Methods of preparing monoclonal andpolyclonal antibodies are routine in the art, as described, for example,in Harlow and Lane, supra, 1988.

The term antibody, as used herein, also encompasses non-naturallyoccurring antibodies and fragments containing, at a minimum, one V_(H)and one V_(L) domain, such as chimeric antibodies, humanized antibodiesand single chain Fv fragments (scFv) that specifically bind c-Met. Suchnon-naturally occurring antibodies can be constructed using solid phasepeptide synthesis, produced recombinantly or obtained, for example, byscreening combinatorial libraries consisting of variable heavy chainsand variable light chains as described by Borrebaeck (Ed.), AntibodyEngineering (Second edition) New York: Oxford University Press (1995). Avariety of useful anti-c-Met monoclonal and polyclonal antibodies arewell known in the art and, in many cases, are commercially available.

The present invention also provides, inter alia, novel methods fordiagnosis and prognosis evaluation for cancer.

In one aspect, the amount of soluble c-Met in a biological sample isdetermined in different patient samples for which either diagnosis orprognosis information is desired, to provide profiles. An profile of aparticular sample is essentially a “fingerprint” of the state of thesample. A normal state may be distinguished from a cancerous state, andwithin cancerous states, different prognosis states (good or poor longterm survival prospects, for example) can be determined. Diagnosis maybe done or confirmed by comparing patient samples with the knownprofiles. By assessing the evolution of c-Met levels at different timesduring disease progression, the stage of disease can be determined aswell as the likely prognosis.

A principle of diagnostic testing is the correlation of the results of aprocedure with particular clinical parameters. The correlationnecessarily involves a comparison between two or more groupsdistinguished by the clinical parameter. A clinical parameter could be,for example, presence or absence of disease, risk of disease, stage ofdisease, severity of disease, class of disease or response to treatmentof disease. Accordingly, the diagnostician uses this correlation toqualify the status of a subject with respect to the clinical parameter.That is, the diagnostician uses the results of a procedure on a subjectto classify or diagnose a subject status with respect to a clinicalparameter, the confidence of the diagnosis/classification being relatedto the classifying or splitting power of the signs or symptoms used inthe test.

The methods described herein for quantifying soluble c-met in abiological sample provides information which can be correlated withpathological conditions, predisposition to disease, therapeuticmonitoring, risk stratification, among others. Although the datagenerated from the methods of this invention is suited for manual reviewand analysis, data processing using high-speed computers can be utilizedas well.

The present methods are particularly useful for diagnosing conditions,evaluating whether certain drugs will have a desired effect, anddetermining prognoses. The present methods can be used for earlydetection of cancer as well as for the optimization of treatmentprotocols and analysis of biopsy samples. The present methods can alsobe used to optimize chemotherapy through assessment of the effect thatthe chemotherapy has on c-Met levels.

In certain embodiments, the invention provides methods for monitoringthe progression of cancer in a patient. The method comprises the stepsof providing a first biological sample from the patient, preferably aurine and/or blood plasma sample, measuring the amount of soluble c-Metin the first biological sample at a first time point, providing a secondbiological sample from the patient, measuring the amount of solublec-Met in the second biological sample at a second time point, anddetermining progression of the disease state in the patient based uponthe change in the amount of soluble c-Met in the biological sample. Incertain embodiments, the amount of soluble c-Met ectodomain in a urineand blood plasma sample will be measured at a first time point and theamount of soluble c-Met ectodomain in a second urine and blood plasmasample will be measured at a second time point and the combinedmeasurement will be used to determine progression of the cancer in thepatient. By measuring the amount of c-Met in a patient sample over time,a clinician will be able to determine whether the cancer has, forexample, regressed and whether the subject has been effectively treated.A clinician can therefore utilize these measurement for tailoringtreatment appropriately. A subject whose cancer has regressed aftertreatment with an anti-cancer agent will have less detectable c-Met thanhe did before the treatment. Similarly, a subject whose cancer hasremained stable during treatment will have similar levels of c-Met as hedid before treatment, and a subject whose cancer has worsened will haveincreased c-Met levels.

In certain embodiments, the present invention provides methods formeasuring the response to cancer therapy comprising the steps ofproviding a first biological sample, preferably a urine and/or bloodplasma sample, measuring the amount of soluble c-Met in the firstbiological sample at a first time point, providing a second biologicalsample from the patient, measuring the amount of soluble c-Met in thesecond biological sample at a second time point, and determiningresponse in the patient based upon the change in the amount of solublec-Met present in the biological sample. In certain embodiments, theamount of soluble c-Met ectodomain in a urine and blood plasma samplewill be measured at a first time point and the amount of soluble c-Metectodomain in a second urine and blood plasma sample will be measured ata second time point and the combined measurement will be used todetermine patient response. The subject may be a positive responder,poor responder, or non-responder. For use herein, a positive responder,is a subject who positively responds to treatment, i.e., a subject whoexperiences success in amelioration of the cancer, including anyobjective or subjective parameter such as abatement; remission;diminishing of symptoms or making the condition more tolerable to thepatient; slowing in the rate of degeneration or decline; making thefinal point of degeneration less debilitating; or improving a subject'sphysical or mental well-being. A positive responder is one in which anytoxic or detrimental side effects of the biologically active agent isoutweighed in clinical terms by therapeutically beneficial effects. Anon-responder is a subject who doesn't respond to the treatment ordoesn't respond to a satisfactory level. A poor responder is a subjectwho responds to treatment but not at the level of the positiveresponder.

In certain embodiments, the cancer therapy will include theadministration of an anti-cancer drug. A clinician or other suitableprofessional can use the information regarding a subject's likelyresponse level to certain anti-cancer drugs to determine an appropriatetreatment regimen for the patient. In certain embodiments, increasingdosages can be provided for those patients that are indicated to be poorresponders or non-responders. In certain embodiments, patients that areindicated to be poor responders, or non-responders will receive adifferent class of drugs or therapy. In certain embodiments, the cancertherapy will be ionizing radiation, hormone ablation therapy, and thelike.

Generally, the amount of soluble c-Met ectodomain present in the sampleof a patient with premalignant cancer will be less than the amount ofsoluble c-Met ectodomain present in the sample of a patient with a lowgrade cancer, and the amount of soluble c-Met ectodomain present in thesample of a patient with a low grade cancer will be less than the amountof soluble c-Met ectodomain present in the sample of a patient with ahigh grade metastatic cancer.

Anti-cancer drugs are known in the art and include, for examplechemotherapeutic agents, alone, or in combination with, radiationtreatment, surgical treatment, or treatments using biological orimmunomodulatory agents. Chemotherapeutic drugs useful in treatingcancer include alkylating agents, antimetabolites, natural products,hormones and antagonists (reviewed in B. A. Chabner and D. L. Longo Eds.Cancer Chemotherapy and Biotherapy, 3rd Edition, 2001). These include,for example, nitrogen mustards, including, for example, mechlorethamine,cyclophosphamide, ifosfamide, melphalan, and chlorambucil; ethyleniminesand methylmelamines, including, for example, hexamethylmelamine andthiotepa; alkyl sulfonates, including, for example, busulfan,carmustine, lomustine, semustine, and streptozocin; triazenes,including, for example, dacarbazine and temozolamide, folic acidanalogs, including, for example, methotrexate and trimetrexate5-fluoropyrimidines including, for example, fluorouracil, floxuridine,ftorafur, capecitabine, and eniluracil, cytidine analogs, includingcytarabine; 5-azacytidine, gemcitabine, purine analogs and relatedinhibitors, including, for example, mercaptopurine, thioguanine,fludarabine, cladribine, and pentostatin; vinca alkaloids, including,for example, vinblastine, and vincristine; taxanes including paclitaxeland docetaxel, topoisomerase II inhibitors, including, for example,etoposide, amsacrine and teniposide; topoisomerase I targeting agentsincluding, including, for example, camptothecin, topotecan, irinotecan,and karenitecin, antibiotics, including, for example, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicamycin, and mitomycin;enzymes, including, for example, L-Asparaginase; biological responsemodifiers, including, for example, IL-2, interferon-alfa IL-1, IL-2,IL-4, IL-12, tumor necrosis factor and macrophage colony stimulatingfactor, platinum coordination complexes, including, for example,cisplatin, oxaloplatin, and carboplatin; anthracenediones, including,for example, mitoxantrone; thalidomide and derivatives including, forexample, revemid, proteosome inhibitors including, for example,bortezomib, substituted ureas, including, for example, hydroxyurea;methylhydrazine derivatives, including, for example, procarbazine;adrenocortical suppressants, including, for example, mitotane andaminoglutethimide; adrenocorticosteroids, including, for example,prednisone; progestins and dexamethasone, including, for example,hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrolacetate; estrogens, including, for example, diethylstilbestrol andethinyl estradiol; antiestrogens, including, for example, tamoxifen;androgens, including, for example, testosterone propionate andfluoxymesterone; antiandrogens, including, for example, flutamide; andgonadotropin releasing hormone analogs, including, for example,leuprolide, aromatase inhibitors including, for example, anastrazole(brand name Arimidex®), exemestane (brand name Aromasin®), and letrozole(brand name Femara®); antibodies directed against cell surface moleculesincluding, for example, rituximab, trastuzumab, CAMPATH, cetuximab andbevacizumab, including antibodies conjugated to toxins, including, forexample, gemtuzumab, and antibodies conjugated to radioisotopesincluding, for example, ibritumomab; anti-cancer antibodies that havebeen humanized to avoid the development of human antimouse antibodies;small molecule tyrosine kinase inhibitors including, for example,gleevec and iressa (reviewed in Noble et al., Science. 2004;303:1800-5); faranesyl transferase inhibitors including, for example,R115777 (tipifarnib, Zarnestra®), SCH66336 (lonafarnib, Sarasar®) andBMS-214662, including formulations of chemotherapy drugs including, forexample, liposomal formulations, including arsenic trioxide, includingcancer differentiating agents including, for example, all trans retinoicacid, including cancer treatments of any kind that are expected toreduce tumor growth, tumor invasiveness, tumor metastasis or overalltumor burden. Methods of administering chemotherapeutic agents fortreating cancer are known in the art. (Goodman and Gilman's ThePharmacological Basis of Therapeutics, Ninth Edition).

The term “cancer therapy” refers to cancer treatment of any kindexpected to reduce tumor growth rate, tumor invasiveness, tumormetastasis, or overall tumor burden, including for example, theadministration of anti-cancer agents, ionizing radiation therapy,hormone ablation therapy, surgical intervention, and the like.

The invention provides a number of methods, reagents, and compounds thatcan be used for the diagnosis and prognosis of cancer. It is to beunderstood that this invention is not limited to particular methods,reagents, compounds, compositions, or biological systems, which can, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting. As used in this specification and theappended claims, the singular forms “a”, “an”, and “the” include pluralreferents unless the content clearly dictates otherwise.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein.

EXAMPLES

Reagents and Cell Culture

Full length purified recombinant human hepatocyte growth factor (HGF)protein was obtained from R&D Systems (294-HG, Minneapolis, Minn.).Antibodies against c-Met were obtained from R&D Systems (Minneapolis,Minn.) or Upstate Biotechnology (DO-24 anti-c-Met mAb; Lake Placid,N.Y.) as noted below.

The following human normal/tumor and primary tumor/metastasis cell linepairs derived from single individuals were obtained from ATCC andmaintained according to ATCC recommendations: CRL7636 (normal skin) andCRL7637 (skin melanoma), HTB125 (normal mammary gland) and HTB126(mammary gland ductal carcinoma), and CCL228 (colorectal adenocarcinoma)and CCL227 (lymph node metastasis of colorectal adenocarcinoma). Thehuman cell line pair UOK124 (renal cell carcinoma) and UOK124 LN (lymphnode metastasis of renal cell carcinoma), as well as UOK261 (bladdercancer) were developed at the Urologic Oncology Branch, National CancerInstitute, National Institutes of Health, Bethesda, Md. 20892 USA;Athauda et al., Clin. Cancer Res. 12: 4154-4162, 2006). UOK124, UOK124LN, UOK261, A431 (epidermoid carcinoma), U-87 MG (glioblastoma), C100(breast carcinoma), H1.177 (C100 transfected with Nm23), PC3 (prostatecarcinoma), PC3M (PC3 derived metastatic variant) and PC3M MxA (PC3Mtransfected with MxA) cells were maintained in DMEM supplemented with10% fetal bovine serum, antibiotics and antimycotics. B5/589 humanmammary epithelial cells were cultured as previously described (Stahl etal., Biochem J 1997; 326:763-72). The MCF-10A derived breast epithelialcell lines 141-M4 (MCF-10A or M1, MCF10AT1k.cl2 or M2, MCF-10CA1h or M3and MCF-10CA1a.cl1 or M4) (Santner et al., Breast Cancer Res Treat 2001;65:101-10) were obtained from the Barbara Ann Karmanos Cancer Institute(Detroit, Mich.) and were maintained in DMEM/F-12 supplemented with 5%horse serum, 0.029M sodium bicarbonate, 10 mM HEPES, penicillin andstreptomycin at 37° C. and 5% CO₂. This culture medium was supplementedwith 10 μg/ml insulin, 20 ng/ml EGF, 0.5 μg/ml hydrocortisone and 100ng/ml cholera toxin for the M1 and M2 cell lines.

SDS-PAGE and Immunoblotting

Analysis of cellular and soluble c-Met by immunoprecipitation andimmunoblotting was performed as described previously (Stahl et al.,Biochem J 1997; 326:763-72). Samples for soluble c-Met analysis wereobtained by harvesting conditioned media (cell culture supernatants)prior to detergent extraction; conditioned media was subjected to highspeed centrifugation and 0.2 micron filtration to remove cells anddebris. To obtain cellular c-Met samples, intact cells were serumdeprived for 16 or 24 h as noted, lysed in cold buffer containingnon-ionic detergents and protease and phosphatase inhibitors and clearedby high speed centrifugation. After immunoprecipitation of clarifieddetergent extracts for 2 h on ice, immunocomplexes were captured usingimmobilized protein-G (GammaBind G-Agarose, Pharmacia), washed, elutedwith SDS sample buffer and subjected to SDS-PAGE and electrophoretictransfer to PVDF membranes (Immobilon P, Millipore). Immunodetection wasperformed by conventional methods using enhanced chemiluminescence (ECLPlus, Amersham).

Electrochemiluminescence Immunoassays

Streptavidin coated 96-well plates designed specifically for use in aMeso Scale Discovery (MSD) Sector 2400 Imager were first blocked withI-Block solution (300 μl/well, 1 h; Applied Biosystems catalog #A1300)that had been prepared according to the manufacturers instructions.Wells were then washed 3 times with PBS (150 μl/well).

For c-Met ectodomain assays, a biotin-tagged, affinity purified c-Metectodomain specific capture antibody (R&D BAF 358 diluted in 0.5% BSA inPBS) was added to each well (5 ug/ml, 25 μl/well) for 1 h with shaking.Wells were washed 3 times with PBS before adding samples or standards(R&D 358-MT recombinant c-Met ectodomain-IgG-Fc fusion protein inPBS+0.5% BSA). Standards (100 μl/well) were added to generate a curvefrom 0.01 ng/ml to 100 ng/ml in semi-log increments for 1 h withshaking. Samples for soluble c-Met analysis were obtained by harvestingconditioned media (cell culture supernatants) prior to detergentextraction; conditioned media was subjected to high speed centrifugationand 0.22 micron filtration to remove cells and debris, then stored at−80° C. prior to c-Met quantitation. In some cases conditioned media wasconcentrated using Centricon YM-10 microconcentration units (Millipore,Inc.) prior to analysis, as described in the text, or subjected to asingle round of immunodepletion using the human c-Met ectodomainspecific monoclonal antibody DO24 (Upstate Biotechnology) followed byantibody capture with protein G agarose. Wells were washed 3 times withPBS before adding detection antibody (R&D AF 276 labeled with MSDSulfotag diluted in 0.5% BSA in PBS) at 1 ug/ml, 25 μl/well for 1 h withshaking. Wells were then washed 4 times with PBS before adding MSD ReadBuffer T with surfactant (150 μl/well) and then read immediately in aMSD Sector 2400 Imager.

For cellular c-Met assays, intact cells at 80% confluence were serumdeprived for 24 h, washed twice with cold PBS and then lysed in coldbuffer containing non-ionic detergents and protease and phosphataseinhibitors. Detergent extracts were clarified by high speedcentrifugation and applied to 96-well plates as described for solublec-Met ectodomain samples, above.

c-Met assays can be performed on biological sample, e.g., biologicaltissue or biological fluid from a patient or human patient. Thebiological tissue can be assayed for c-Met ectodomain as a detergentextract of the tissue. Biological tissue from a patient can be extractedwith non-ionic detergent buffer solutions as described herein forpreparing cell extracts. The tissue can be minced or ground (at 4° C. oron ice) to maximize surface area available for extraction, and generallythe extraction (also on ice) is performed for a much longer period (2-3hours) as opposed to a few minutes for cultured cells. See, for example,U.S. Pat. No. 5,674,753.

For HGF immunoassays, I-Block treated plates were coated as describedabove with an affinity purified HGF specific capture antibody (R&D MAB694 diluted in 0.5% BSA in PBS) that had been biotin labeled. Wells werewashed as described above before adding samples or standards (R&D 294-HGrecombinant HGF protein in PBS+0.5% BSA); standards were added togenerate a curve from 0.03 ng/ml to 30 ng/ml in semi-log increments.Wells were washed before adding detection antibody (R&D AF-294 labeledwith MSD Sulfotag diluted in 0.5% BSA in PBS) at 1 ug/ml, 25 μl/well.Wells were then washed 4 times with PBS before adding Read Buffer T andreading in a MSD Sector 2400 Imager.

All samples were measured in quadruplicate unless otherwise noted. Meanvalues from negative control wells were subtracted from all other rawvalues and a standard curve was then constructed by plotting signalintensity against 358-MT c-Met Fc fusion protein or recombinant HGFprotein standard concentration. A non-linear regression curve fittingalgorithm (Microsoft Excel or GraphPad Prism software) was used togenerate an equation from which sample values for c-Met or HGFconcentration were derived from mean signal intensity values. Meanvalues among groups were compared for statistically significantdifferences using unpaired t-test (paired human cell lines) or analysisof variance (ANOVA; MCF10A derived cell lines); R² and P values arepresented in the text and figure legends.

Human Tumor Xenografts in Mice

Subconfluent UOK261 and U-87 MG cells were trypsinized, washed two timesin PBS to remove serum and then resuspended in HBSS at a concentrationof 1×10⁷ cells/ml. One hundred microliters containing 1×10⁶ cells wereinjected subcutaneously into the right flank of 12-week-old maleSCID/beige mice (Taconic Inc., Germantown, N.Y.). Tumor growth wasmonitored weekly by caliper measurements and tumor volume was calculatedbased on the formula (length×length×width)/6. Blood was obtained via thetail vein or retromandibular perforation; samples were collected in thepresence of citrate as an anticoagulant were centrifuged at 1500 rpm, 4°C. to remove cells and plasma samples were stored at −80° C. prior toanalysis. At each of three weekly time points 4 mice were sampled pergroup. Samples were analyzed for soluble c-Met using theelectrochemiluminescence immunoassay described above; values shownrepresent the mean of triplicate measurements. In FIG. 6, standarddeviations are smaller than the symbol size in all cases. Mouse urinesample pH was adjusted to pH 7.5 with Trizma-HCl, 2M, pH 8.0 (Sigma),then centrifuged at 1500 rpm for 5 minutes to remove cells and debris.Pooled SCID/beige normal mouse plasma was used as a diluent for standardcurves and was obtained in bulk from Taconic Inc. Pooled urine fromnormal SCID/beige mice was pH-corrected and used as a diluent forstandard curves run in assays to measure c-Met concentration in mouseurine samples. R² and P values from linear regression analysis of mousedata are presented in the text and figure legends.

c-Met Shedding by Cultured Cells: Characterization and Assay Development

c-Met ectodomain shedding was examined in a cultured cell model ofbreast cancer progression where successive derivatives of the parentcell line show increasing malignancy (Santner et al., Breast Cancer ResTreat 2001; 65:101-10). MCF-10A (M1) is a spontaneously immortalizednormal breast epithelial cell line which was transfected with activatedHras and xenografted in mice to obtain the premalignant MCF10AT1k.cl2(M2) cell line. Subsequent passages in mice and single cell cloningfacilitated the isolation of cell lines that produced tumors with thephenotypic characteristics a low grade carcinoma (MCF-10CA1h or M3) anda high grade metastatic carcinoma (MCF-10CA1a.cl1 or M4). In contrast tomany carcinoma derived cell lines where c-Met is overexpressed, cellsurface c-Met expression among the four cultured cell lines appeared todecrease with increasingly malignant phenotype (FIG. 1A). Nonetheless,analysis of c-Met shedding over 16 h by immunoblotting withectodomain-specific monoclonal antibodies showed progressively higherectodomain levels from normal cells to those displaying a metastaticphenotype (FIG. 1B). c-Met ectodomain fragments of approximate molecularmasses 75, 85 and 100 kDa were the predominant species observed, similarto those observed in the normal human mammary epithelial cell lineB5/589 (FIG. 1B) and to the predominant species present in cell culturesupernatants and human plasma as reported previously [3-7]. These c-Metreactive protein bands were detectable with several antibodies againstthe c-Met ectodomain, but not with polyclonal antisera raised against apeptide corresponding to the carboxyl terminal c-Met sequence (data notshown).

To better characterize c-Met shedding across a range of cell lines andbiological samples, a two site immunoassay was developed to providegreater sensitivity, higher throughput and more precise quantitationthan immunoblotting. Distinct human c-Met specific antibodies are usedfor capture and detection; capture antibodies were biotin labeled foruse with streptavidin coated multiwell plates and the amount ofdetection antibody bound was measured by electrochemiluminescencegenerated using ruthenium chelates in the presence of sacrificial redoxco-reactants. The optimized c-Met immunoassay has a threshold ofdetection of 750 fg for the purified recombinant c-Met ectodomainprotein and a dynamic range of four log units (FIG. 2A). Samplequenching or hypersensitivity that could occur in complex biologicalsamples was investigated by adding known amounts of recombinant c-Metectodomain to cultured cell supernatants from B5/589 mammary epithelialcells (data not shown) or A431 epidermoid carcinoma cells (FIG. 2B),both of which contained shed c-Met ectodomain as determined byimmunoblotting. Agreement between expected and observed c-Metconcentrations indicated the absence of any interference (FIG. 2B).Ultrafiltration of B5/589 conditioned medium through a membrane with a10 kDa cutoff removed all of the c-Met signal, consistent with theectodomain molecular masses observed by immunoblotting (FIG. 2C). Thec-Met selectivity of the immunoassay was tested by subjecting B5/589conditioned medium to immunodepletion using a monoclonal antibodyagainst c-Met ectodomain (DO-24) that was distinct from either captureor detection antibody used in the immunoassay. As shown in FIG. 2C, asingle round of immunodepletion resulted in an 80% loss of signal.

To determine whether shed c-Met ectodomain fragments could act ascompetitive antagonists of ligand binding by cell surface receptors, thetwo site c-Met immunoassay was adapted to analyze the HGF bindingcapacity of c-Met fragments cultured cells conditioned medium. Apurified recombinant full length c-Met ectodomain-IgG Fc fusion proteinwas used as positive control for HGF binding. The amount of c-Metectodomain present in B5/589 conditioned medium was determined from ac-Met-Fc standard curve and assays were designed such that equal amountsof c-Met ectodomain in B5/589 conditioned medium and in the c-Met Fcfusion protein preparation were captured in replicate wells. Purifiedrecombinant HGF was added to two sets of wells at concentrations of 10,100 and 300 ng/ml; one set of wells was then detected using anti-c-Metand the other with anti-HGF. An HGF standard curve was created using thesame HGF detection antibody and an HGF-specific capture antibody. Nochange in c-Met detection was observed in c-Met Fc containing wells thathad bound HGF, indicating that bound HGF did not interfere with c-Metcapture or detection (data not shown). As shown in FIG. 2D, the c-Met Fcfusion protein bound HGF in direct proportion to the amount added,whereas no HGF binding was detected for the c-Met fragment present inB5/589 conditioned medium. These results, consistent with prior studies(Wajih et al., Circ Res 2002; 90:46-52), show that the predominant c-Metectodomain fragments found in B5/589 cell culture supernatants have lostmeaningful HGF binding capability. In light of the observation that theB5/589 c-Met ectodomain fragments are very similar in size anddistribution to those shed by a variety of cultured cell lines as wellas to what has been found in human plasma (Wajih et al., Circ Res 2002;90:46-52), c-Met shedding does not yield a soluble receptor fragmentthat could compete efficiently for ligand binding by intact cellularc-Met.

c-Met Shedding Correlates with Malignancy in Cultured Cell Cancer Models

Quantitative analysis of c-Met shedding by the MCF 10A derived breastcancer cell lines that were analyzed previously by immunoblotting showedexcellent agreement in the trends among the cell lines for both cellularc-Met expression and c-Met ectodomain shedding (FIG. 1B and FIG. 3,upper panels). The external recombinant protein standard in theimmunoassay allows results to be expressed in absolute terms, e.g.receptor concentration or number per total cellular protein or per cell.This enables realistic comparisons between successive experiments,different cell lines or with other biological samples. Manipulation ofthe numerical results also offers insight into trends in sheddingrelevant to molecular mechanism, such as determining shedding rate percellular receptor (FIG. 3, 3^(rd) panel) or the percentage of cellularreceptor shed per time interval (FIG. 3, bottom panel). In the MCF10Aderived model of breast cancer progression, the steady state cellularc-Met expression level is progressively and significantly lower at eachstep of increasingly malignant phenotype (ANOVA R²=0.998; P<0.0001). Incontrast, c-Met shedding is significantly increased with the change frompremalignant (M2) to malignant (M3) phenotype (FIG. 3, 2^(nd) panel,t-test P<0.001). This trend is maintained in the metastatic (M4) cellline (FIG. 3, 2^(nd) and 3^(rd) panels) and shedding in M4 is nearlyfour-fold higher than its normal counterpart M1 when expressed as afunction of available cellular c-Met (FIG. 3, bottom panel; 4 groupANOVA R²>0.995; P<0.0001). The observed trends in expression andshedding are consistent with significant and progressive increases inproteolytic activity characteristic of advancing breast cancer.

Cellular and soluble c-Met levels in a series of paired human celllines, each obtained from a single cancer patient, derived from tumorvs. corresponding normal tissue or, primary tumor vs. distant metastaticlesion were measured. Specifically, the paired cell lines were derivedfrom normal skin (CRL7636) and skin melanoma, (CRL7637), normal mammarygland (HTB125) and mammary gland ductal carcinoma (HTB126), colorectaladenocarcinoma (CCL228) and lymph node metastasis of colorectaladenocarcinoma (CCL227), and renal cell carcinoma (UOK124) and lymphnode metastasis of renal cell carcinoma (UOK124 LN).

All four cell line pairs exhibited one overall trend similar to thatdisplayed by the MCF10A model: significantly greater c-Met shedding ratewith more malignant phenotype (FIG. 4, 2^(nd), 3^(rd), and 4^(th)panels). In contrast, cellular c-Met levels varied widely among thepaired lines (FIG. 4, top panel). Relative to the normal skin derivedcell line CRL7636, cellular c-Met was 60% lower then in the pairedmelanoma derived cell line CRL7637 (top panel), while soluble c-Metconcentration was 60% greater (2^(nd) panel), contributing to a 4-foldgreater c-Met shedding rate per cell protein (3^(rd) panel) and perreceptor (4^(th) panel) correlating with the acquisition of malignancy.These findings suggest that, similar to the MCF10A derived cell lines,significantly increased proteolytic activity was the primary cause ofthe observed trend. In contrast, in the cell line pair derived fromnormal mammary gland and ductal carcinoma (HTB125 and HTB126),acquisition of malignancy correlated with 2.5 fold greater cellularc-Met level (top panel) and 4-fold greater soluble c-Met level (2^(nd)panel) producing a 2.5 fold greater c-Met shedding rate per cell protein(3^(rd) panel). This trend represented only a modest increase in thepercentage of cellular c-Met shed in 24 h (bottom panel), suggestingthat unlike the MCF10A derived breast cancer model, receptoroverexpression contributed significantly to the increased receptorshedding observed with malignant transformation. This difference inunderlying mechanism of increased c-Met shedding between two breastcancer cell models could reflect the oncogenic heterogeneity in breastcancer that is becoming more broadly appreciated.

Similar in trend to HTB125 and HTB126, but remarkable in magnitude, thecell line derived from a lymph node metastasis of colorectal carcinoma(CCL227) showed 40-fold greater cellular c-Met expression relative tothe primary tumor derived cell line (CCL228) and 75-fold greater rate ofc-Met shedding per total cell protein (FIG. 4, upper two panels).Correcting these values for receptor protein yielded a 2.7 fold greatershedding rate in the metastasis-derived line relative to the primarytumor line (FIG. 4, 3^(rd) panel). The renal cell carcinoma derived cellline UOK124 and corresponding lymph node metastasis derived line UOK124LN showed little difference in cellular c-Met expression (top panel),while soluble c-Met level was 60% greater in UOK124 LN (2nd panel),contributing to a 3-fold greater c-Met shedding rate per cellularreceptor (3^(rd) panel) and a doubling of the percentage of cellularreceptor shed in 24 h (bottom panel). Thus, similar to the trendsobserved for the MCF10A derived breast carcinoma model and the skinmelanoma cell line pair, significantly greater proteolytic activity waslikely to be responsible for the greater c-Met shedding rate thatcorrelated with increased malignancy.

In addition to cultured normal/tumor or tumor/metastasis cell linepairs, cellular and soluble c-Met levels in two genetically engineeredmodels representing reconstitution of metastasis suppressor genes inaggressively malignant prostate and breast tumor derived cell lines wereexamined. The prostate cancer cell line PC3 is tumorigenic in mice butnot metastatic; PC3M is a PC3 derived cell line that is aggressivelytumorigenic and metastatic (Chu et al., Cancer Genet Cytogenet 2001;127:161-7.) This phenotypic difference was exploited to identify geneswhose loss could contribute to metastasis in prostate cancer, leading tothe identification of the MxA gene as a suppressor of metastasis (Aebiet al., Mol Cell Biol 1989; 9:5062-72 and unpublished observations).Upon reconstitution of MxA expression in PC3M, the aggressive metastaticphenotype of cultured cell xenografts in mice is reverted to that of theparental cell line (unpublished observations). Cellular c-Met expressionin PC3M was nearly double that of PC3; restoration of MxA expression wasassociated with an even greater cellular c-Met expression level (FIG.5A, top panel). Shedding by PC3M was 50% greater than that of theparental cell line, consistent with the overall trend of increasedshedding with increasing malignancy observed in other cell models (FIG.5A, 2^(nd) panel), and apparently driven by increased c-Met expression.While the absolute level of c-Met shedding in PC3M and PC3M MxAtransfectants was similar, a lower the rate of c-Met shedding percellular receptor was detected in MxA transfectants (FIG. 5A, 3^(rd) and4^(th) panels). These results suggest that MxA expression had nosignificant effect on c-Met expression, but suppressed the sheddingmechanism to a level comparable to that displayed by the non-metastaticPC3 cell line.

In contrast to the PC3 based model of prostate cancer metastasis, adramatic effect on cellular c-Met expression was exhibited by themetastasis suppressor gene Nm23 in a genetically engineered cell modelof breast cancer progression (FIG. 5B). In this model, the breast cancercell line C100, which aggressively forms primary tumors and metastasesin mouse xenografts, was reverted to a non-metastatic phenotype upontransfection with Nm23 (H1.177) (Leone et al., Oncogene 1993;8:2325-33]. Correlated with the loss of metastatic phenotype was 70%lower cellular c-Met expression in H1.177 relative to the parental cellline (FIG. 5B, top panel) and 75% lower soluble c-Met production (FIG.5B, 2^(nd) panel). A 20% lower level of c-Met shedding per cellularreceptor was also associated with Nm23 expression (FIG. 5B, 3^(rd) and4^(th) panels), suggesting that shedding was attenuated primarilythrough the suppression of c-Met expression by Nm23.

c-Met Shedding Correlates with Tumor Burden in Tumor Xenograft MouseModels

It was observed that cultured tumor cells expressing c-Met tended toshed more c-Met ectodomain than their normal tissue counterparts,independent of changes in overall c-Met expression level, and that thistendency was enhanced with increasingly malignant phenotype. Twodifferent human tumor cell lines were injected subcutaneously with knownc-Met shedding rates (data not shown) into immunocompromised(SCID/Beige) mice and thereafter measured tumor volume and plasmasoluble c-Met levels at weekly intervals. The cell line UOK261 wasderived from a human bladder carcinoma and displayed a relatively highlevel of soluble c-Met in culture, whereas the cell line U-87 MG wasderived from a human glioblastoma and displayed a lower level of c-Metshedding in culture. c-Met signaling is suspected of playing animportant role in the progression of both cancers represented by thesemodels [Burgess et al., Cancer Res 2006; 66: In Press; Rosen et al., JUrol 1997; 157:72-8; Cheng et al., J Clin Oncol 2002; 20:1544-50.) Notethat the antibodies used in the immunoassay described here do notcross-react with mouse c-Met, thus the assay was conducted in theabsence of any normal soluble murine c-Met background; any c-Metdetected originated from the human tumor xenografts. Pooled plasmasamples obtained from the same strain of mice was used as a diluent forthe recombinant c-Met ectodomain-Fc standard curve, so that the absolutesoluble c-Met values obtained could be related directly to thoseobtained from cultured cell experiments, as well as to future animalstudies.

Remarkably, soluble human c-Met was easily detected in plasma samplesobtained from several mice receiving UOK261 xenografts more than a weekbefore subcutaneous tumors became palpable (data not shown).Non-palpable tumors up 5.0 mm³ in volume are difficult to detectradiologically and represent an early but nonetheless clinicallyrelevant stage of tumorigenesis. The soluble c-Met fragments measured inthe immunoassay were similar in size to the fragments found in culturedcell conditioned media, as determined by SDS-PAGE and immunoblotting(data not shown). With increasing tumor mass, each mouse showedsignificant weekly increases in plasma c-Met levels (P<0.05; data notshown); these data were pooled and plotted as plasma c-Met against tumorvolume (FIG. 6, left upper panel). Regression analysis demonstrated adirect linear relationship between circulating soluble c-Metconcentration and tumor burden (FIG. 6, left upper panel, R²=0.944; n=4animals). Consistent with the lower level of c-Met shedding by culturedU-87 MG cells, soluble c-Met was not detected in plasma samples frommice receiving the U-87 MG glioblastoma xenografts before the tumorsbecame palpable. Nonetheless, the smallest measurable tumors were eachassociated with plasma c-Met levels that were well above the thresholdof detection, and each mouse showed significantly increasing c-Metconcentrations measured at weekly intervals (P<0.05; data not shown).Regression analysis of the pooled data plotted as c-Met concentrationagainst tumor volume also supported a direct linear soluble c-Met/tumorburden relationship (FIG. 6, right upper panel, R²=0.933; n=4 animals).

As shown in the left lower panel of FIG. 6, subcutaneous UOK261xenografts produced urinary soluble c-Met concentrations that weredetectable at very small tumor sizes and that increased linearly withtumor burden over weekly intervals (R²=0.922, P<0.01, n=4 animals).Urinary soluble c-Met concentrations were typically less that1/1000^(th) of corresponding plasma c-Met concentrations (FIG. 6, leftlower panel). Urine samples from mice receiving U-87 MG xenografts alsocontained soluble c-Met at early stages of tumor growth that increasedsignificantly with time in each mouse (P<0.05; data not shown), andgroup data showed a similar direct linear relationship between c-Metconcentration and overall tumor burden (FIG. 6, right lower panel;R²=0.953, n=4 animals).

These results demonstrate that soluble c-Met produced by tumor derivedcultured cell xenografts can be detected in plasma at very early stagesof tumorigenesis, and that urinary c-Met concentration is a reliableindicator of the soluble c-Met level in plasma. These data furthersupport the overall fidelity of c-Met shedding as an index of malignantphenotype, tumor progression and tumor burden in c-Met expressing modelsof oncogenesis. The simplicity and sensitivity of the assay describedhere make it amenable to high throughput screening.

c-Met Shedding in a Group of Normal Patients and Patients with CancersDelineated in FIGS. 7-9.

Soluble c-Met was measured in urine samples as described in for mouseurine, with the exception that the standard curve was generated inphosphate buffered normal saline, not pooled mouse urine. Urinecreatinine was measured using a standard, commercially availablespectrophotometric method which is routinely used in medicallaboratories. Samples for all assays were performed in triplicate orquadruplicate. Prior to measurements, samples were centrifuged to removecells and debris, filtered through 0.22 micron filters, and the pH wasadjusted to 7.4.

c-Met Shedding in a Group of Normal Patients and Patients with BladderCancer.

Soluble c-Met was measured in urine samples as described in for mouseurine, with the exception that the standard curve was generated inphosphate buffered normal saline, not pooled mouse urine. Urinecreatinine was measured using a standard, commercially availablespectrophotometric method which is routinely used in medicallaboratories. Samples for all assays were performed in triplicate orquadruplicate. Prior to measurements, samples were centrifuged to removecells and debris, filtered through 0.22 micron filters, and the pH wasadjusted to 7.4. Values of soluble c-Met were obtained from the raw datausing the standard curve (run in every assay); the standard curve wasgenerated as described, final values are expressed as pg/ml. Urinecreatinine was also measured against a standard curve and values areexpressed as mg/ml. Corrected c-Met/creatinine values are expressed aspg/mg. Statistical analysis was performed using GraphPad Prism software,using assumptions and conventions accepted throughout the researchcommunity. t-test at the p<0.05 level is the standard method forcomparing the mean value obtained from patient replicates to the normalgroup mean, i.e. determining if any difference is unlikely to be due tochance. See FIG. 12.

Normals were healthy adult volunteers with no medical history of cancerand asymptomatic for diseases of the kidney, prostate, bladder orurinary tract at the time the sample was obtained, as established byroutine medical laboratory tests, physical exam, and interview.

Bladder cancer samples were obtained from adult patients with biopsyproven (graded and staged by a licensed pathologist) bladder cancer orTCC (transitional cell carcinoma, which originates in ureteralepithelium, generally classified with bladder cancer). Patients hadactive or stable disease and samples were obtained prior to any surgicalintervention.

Table 1 below provides the soluble c-Met measurements as pg per mg urinecreatinine.

TABLE 1 Statistics Normals Bladder Cancer Number of values 30 12 Minimum892.3 1498 25% Percentile 3071 4343 Median 4344 6658 75% Percentile 571913990 Maximum 7742 33090 Mean 4193 10600 Standard Deviation 1835 9278Standard Error 335 2678 Lower 95% Confidence 3508 4709 Interval Higher95% Confidence 4878 16500 Interval KS Distance 0.09099 0.2363 P ValueP > 0.10 P > 0.10 Passed Normality Test Yes Yes (* = 0.05) P ValueSummary ns ns Coefficient of Variation 43.76% 87.49% Geometric mean 36897644Adaptation of the Soluble c-Met Immunoassay for Analysis of Human PlasmaSamples.

Initial measurements of soluble c-Met in human plasma samples obtainedfrom normal healthy volunteers, using the recombinant 358-MT c-Met-Igfusion protein (R&D Systems) as standard and the same assay protocolused for cultured cell supernatants, revealed very high soluble c-Metlevels were present.

Experiments confirm that the measurements were reliable and not due to afactor present in plasma other than bona fide c-Met protein:

1. Samples of goat plasma and mouse plasma were measured to determinewhether a plasma constituent other than c-Met might contribute to signalintensity. Because the assay reagents do not cross-react with c-Metprotein from mouse or goat, any reading above background would begrounds to suspect a contributory factor in human plasma other thanc-Met. Neither goat nor mouse samples displayed soluble c-Met readingabove background.

2. Samples of human plasma were diluted with three different buffersover a four log range and then measured in the assay. In many cases,artifactual readings do not decrease linearly with dilution. UsingTris-HCl, PBS or de-ionized water, the signal present in human plasmadirectly in proportion to dilution factor.

3. Samples of human plasma were subjected to denaturing conditions(heating to 95° C. for 30 minutes) prior to measurement of soluble c-Metin that assay. Under these conditions, many plasma proteins areprecipitated. Samples subjected to this protocol did not show c-Metreadings above background.

4. Samples of human plasma were subjected to immunodepletion of c-Metprior to measurement in the assay. Ideally, repeated rounds ofimmunodepletion should remove soluble c-Met from the sample, reducingthe measured value to background levels. The protocol used and resultsobtained from a representative experiment are shown below. To controlfor the non-specific removal of c-Met from the sample by the protein Gbeads used to capture immunocomplexes, a mock immunodepletion wasperformed on human plasma samples in parallel which using a goat-derivedIgG directed against a protein not present in human plasma.

5. Samples of human plasma subjected to immunodepletion were analyzed bySDS-PAGE and immunoblotting with another anti-c-Met antibody not used inthe immunoassay. A standard curve of 358-MT samples were run in parallelto estimate the amount of soluble c-Met present. Results were consistentwith the immunodepletion experiments shown below (data not shown). Crudeestimates of soluble c-Met in human plasma based on immunoblotting werein the range of 100 ng/ml.

Protocol for Immunodepletion (ID) of Soluble c-Met from Human Plasma:

1. To 5 ml human plasma add anti-c-Met R&D AF276 (1 μg/ml of plasma) andincubate on ice for 1 hour; agitate gently every 15 min. In parallel forMOCK samples, add Goat IgG (Rockland) at the same concentration. Repeatall subsequent steps with both ID and MOCK samples.

2. Add Protein G-Sepharose (Amersham; 30 μl/ml plasma) and rotate at 4 Cfor 1 hour.

3. Spin at 3000 rpm at 4° C. 1-2 minutes in Eppendorf desktop centrifugeto pellet beads. Remove supernatant. Take sample from supernatant (storeon ice) and repeat steps 1-3 three more times for a total of fourimmunodepletion steps.

In FIG. 13, the upper panel shows rounds 1 through 4 of immunodepletionon the category (x) axis, and signal strength in the assay on theY-axis. The undepleted plasma samples value is shown as a blue bar nextto each round of immunodepletion. Signal values are listed numericallyabove each bar. Background value is approximately 1500 units. The bottompanel shows the immunodepletion samples alone to better visualize thedegree of depletion. Variation between triplicate samples is consistentless than 2%.

The results of all experiments support the conclusion that 100% of thesignal generated by assay of soluble c-Met in plasma represent bona fidec-Met protein. Measurement of soluble c-Met protein in human plasmasamples obtained from a small set of healthy volunteers suggests thatthe normal concentration range is approximately 100 ng/ml. Analysis of alarger sample set is underway. Given the sensitivity of the assay andthe initial concentration estimate, it should be possible to diluteplasma samples 100-fold prior to measurement, using approximately 100microliters of final volume per well. This should allow replicates of 4to 6 wells/sample to be generated from very small (10 microliter) plasmasamples.

From the foregoing description, various modifications and changes in thecompositions and methods will occur to those skilled in the art. Allsuch modifications coming within the scope of the appended claims areintended to be included therein. Each recited range includes allcombinations and sub-combinations of ranges, as well as specificnumerals contained therein.

All publications and patent documents cited above are herebyincorporated by reference in their entirety for all purposes to the sameextent as if each were so individually denoted.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and can be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

1. A method for identifying a patient having an increased risk ofdeveloping cancer comprising: determining the concentration of solublec-Met ectodomain in a biological fluid from the patient, and comparingthe concentration of soluble c-Met ectodomain in the biological fluidfrom the patient to the concentration of soluble c-Met ectodomain in acorresponding biological fluid from a control population, wherein anincreased concentration of soluble c-Met ectodomain in the biologicalfluid from the patient relative to that in the biological fluid from thecontrol population indicates that the patient has an increased risk ofdeveloping cancer.
 2. The method of claim 1 wherein the biological fluidis blood plasma or urine.
 3. The method of claim 1 wherein thebiological fluid is urine.
 4. The method of claim 3 wherein a solublec-Met ectodomain level from about 3500 pg c-Met per mg urine creatinineto about 4900 pg c-Met per mg urine creatinine is indicative of anincreased risk of developing cancer in the patient.
 5. The method ofclaim 3 wherein a soluble c-Met ectodomain level of about 3500 pg c-Metper mg urine creatinine or greater is indicative of an increased risk ofdeveloping cancer in the patient.
 6. The method of claim 3 wherein asoluble c-Met ectodomain level of about 4900 pg c-Met per mg urinecreatinine or greater is indicative of an increased risk of developingcancer in the patient.
 7. The method of claim 3 wherein soluble c-Metectodomain level of about 3800 pg c-Met per mg urine creatinine orgreater is indicative of an increased risk of developing cancer in thepatient.
 8. The method of claim 3 wherein a soluble c-Met ectodomainlevel of about 4200 pg c-Met per mg urine creatinine or greater isindicative of an increased risk of developing cancer in the patient. 9.The method of claim 3 wherein a soluble c-Met ectodomain level of about4500 pg c-Met per mg urine creatinine or greater is indicative of anincreased risk of developing cancer in the patient.
 10. The method ofclaim 1 wherein the concentration of soluble c-Met ectodomain in a urinesample and a blood plasma sample from the patient is measured and thecombined measurement is used to determine if the patient has anincreased risk of developing cancer.
 11. The method of claim 1 whereinan increased concentration of soluble c-Met ectodomain in the biologicalfluid from the patient relative to that in the biological fluid from thecontrol population indicates that the patient has an increased risk ofdeveloping metastatic cancer.
 12. The method of claim 1 wherein thecancer is a cancer of the urinary tract.
 13. The method of claim 12wherein the cancer is kidney cancer, renal cancer, bladder cancer, orrenal cell carcinoma.
 14. The method of claim 13 wherein the cancer isbladder cancer.
 15. The method of claim 1 wherein the cancer is prostatecancer.
 16. The method of claim 1 wherein the cancer is breast cancer.17. A method of measuring the concentration of soluble c-Met ectodomainpresent in a biological fluid from a human patient comprising:determining the concentration of soluble c-Met ectodomain present in abiological fluid from the patient, and comparing the concentration ofsoluble c-Met ectodomain present in the biological fluid from thepatient to the concentration of soluble c-Met ectodomain in acorresponding biological fluid from a control population.
 18. The methodof claim 17 wherein the biological fluid is blood plasma or urine. 19.The method of claim 17 wherein the biological fluid is urine.
 20. Themethod of claim 19 wherein a soluble c-Met ectodomain level from about3500 pg c-Met per mg urine creatinine to about 4900 pg c-Met per mgurine creatinine indicates that the patient has an increased risk ofdeveloping cancer.
 21. The method of claim 19 wherein a soluble c-Metectodomain level of about 3500 pg c-Met per mg urine creatinine orgreater indicates that the patient has an increased risk of developingcancer.
 22. The method of claim 19 wherein a soluble c-Met ectodomainlevel of about 4900 pg c-Met per mg urine creatinine or greater isindicative of an increased risk of developing cancer in the patient. 23.The method of claim 19 wherein soluble c-Met ectodomain level of about3800 pg c-Met per mg urine creatinine or greater is indicative of anincreased risk of developing cancer in the patient.
 24. The method ofclaim 19 wherein a soluble c-Met ectodomain level of about 4200 pg c-Metper mg urine creatinine or greater is indicative of an increased risk ofdeveloping cancer in the patient.
 25. The method of claim 19 wherein asoluble c-Met ectodomain level of about 4500 pg c-Met per mg urinecreatinine or greater is indicative of an increased risk of developingcancer in the patient.
 26. The method of claim 17 wherein theconcentration of soluble c-Met ectodomain in a urine sample and a bloodplasma sample from the patient is measured and the combined measurementis used to determine if the patient has an increased risk of developingcancer.
 27. The method of claim 17 wherein an increased concentration ofsoluble c-Met ectodomain in the biological fluid from the patientrelative to that in the biological fluid from the control populationindicates that the patient has an increased risk of developingmetastatic cancer.
 28. The method of claim 17 wherein the cancer is acancer of the urinary tract.
 29. The method of claim 28 wherein thecancer is kidney cancer, renal cancer, bladder cancer, or renal cellcarcinoma.
 30. The method of claim 29 wherein the cancer is bladdercancer.
 31. The method of claim 17 wherein the cancer is prostatecancer.
 32. The method of claim 17 wherein the cancer is breast cancer.